Lung edema is a life-threatening complication of lung injury. The active transepithelial Na+ ion transport performed in part by the alveolar epithelial Na,K-ATPase is critical for lung edema clearance. Edema impairs gas exchange, which leads to alveolar hypoxia. Hypoxia, in turn, impairs intercellular adhesion and decreases the amount of the Na,K-ATPase at the plasma membrane, worsening clinical outcomes. The minimal functional unit of the Na,K-ATPase is a dimer consisting of an and a subunit, which not only transports ions, but also acts as a cell adhesion molecule. FXYD5 is a one of the 7 tissue-specific regulatory subunits of the Na,K- ATPase activity, which is also implicated in the impairment of intercellular junctions. In preliminary experiments, we observed that hypoxia impairs intercellular adhesion and up-regulates FXYD5 in alveolar epithelial cells. We hypothesize that the Na,K-ATPase is important for modulating the integrity of the alveolar epithelium by strengthening cell-cell contacts due to the interaction between the Na,K-ATPase 1 subunits of neighboring alveolar epithelial cells and by weakening these contacts by FXYD5- mediated impairment of 1:1 bridges during hypoxia. In specific aim # 1, we will determine whether the interactions between the Na,K-ATPase 1 subunits of neighboring alveolar epithelial cells are impaired by hypoxia We will determine whether modulating 1:1 interactions by removing N-glycans or modifying their structure alters cell-cell adhesion during normoxic and hypoxic conditions in vitro or in vivo. In specific aim #2, we will determine whether FXYD5 up-regulation during hypoxia impairs 1:1 interactions. By over-expressing or silencing of FXYD5 in lung epithelial cells, in both normal and hypoxic conditions, we will determine whether FXYD5 contributes to hypoxia-induced impairment of intercellular adhesion by disrupting the interaction between the Na,K-ATPase subunits of neighboring cells in vitro or in vivo. In specific aim #3, we will investigate chaperone-assisted maturation pathways of the Na,K-ATPase in the endoplasmic reticulum (ER) of alveolar epithelial cells in normal conditions and during hypoxia. In experiments performed for this proposal, we observed that hypoxia results in significant retention of the Na,K-ATPase in the ER of alveolar epithelial cells and thus decreases its abundance in the plasma membrane, which would impair edema fluid clearance and barrier function. We will determine whether up-regulation of ER chaperones by N-glycan processing inhibitor, castanospermine, rescues the maturation of the Na,K-ATPase and whether application of this inhibitor prior to exposure of mice to hypoxia improves barrier function. The proposed studies will provide insights into non- canonical roles of the Na,K-ATPase in stabilization of cell cell contacts, which are crucial for normal function of alveolar epithelia. Understanding the mechanism(s) underlying chaperone-assisted maturation of the Na,K- ATPase in alveolar epithelial cells and finding the means to prevent ER retention of the enzyme during hypoxia may lead to more effective treatment for pulmonary edema and acute lung injury.